1. Field of the Invention
The invention generally relates to the fields of medicinal chemistry and biochemistry. It concerns novel compounds useful for treatment of diseases related to the Src family of tyrosine kinases, methods of synthesis of these compounds and methods of treatment employing these compounds. The novel compounds are thienopyrimidine-based compounds capable of inhibiting the Src family of tyrosine kinases.
2. Related Art
It is a current problem that there are few potent small-molecule inhibitors of the Src family of tyrosine kinases that possess suitable pharmacokinetics, affinity, or specificity to serve as effective treatments for disease (Zhu et al., 1999; Sun et al., 2000; Missbach et al., 2000). Many previously identified small-molecule inhibitors show low specificity for individual protein tyrosine kinases (PTKs) and/or require high concentration of the compound to inhibit the kinase.
The thienopyrimidine-based inhibitors of the Src family act by blocking the enzymatic activity of some or all members of the Src family. Src is a protein tyrosine kinase (PTK) associated with cellular membranes and is involved in signal transduction and growth regulation pathways. It transmits cellular signals by transferring the gamma phosphate of ATP to the side chain of tyrosine residues on substrate proteins. To this date, nine members of the Src protein tyrosine kinase family have been discovered. The members are Src, Yes, Fyn, Fgr, Blk, Lck, Lyn, and Hck. Fgr, Blk, Lck, Lyn, and Hck are expressed and active primarily in hematopoietic cells (Wiener et al., 1999). Alterations in the phosphorylation of Src substrates are key events in cellular signaling. Most normal cells contain very low levels and activity of Src (Barnekow, 1989; Punt et al., 1989) and the enzyme is not required for the establishment or maintenance of cell viability (Soriano et al, 1991).
Src activity is greatly increased in many human cancers: breast cancer (Ottenhoff-Kalff et al., 1992; Partanen, 1994), stomach cancer (Takeshima et al., 1991), colon cancer (Rosen et al., 1986; Bolen et al., 1985; Bolen et al., 1987; Cartwright et al., 1989; Cartwright et al., 1990; Talamonti et al., 1992; Talamonti et al., 1993; Termuhlen et al., 1993), hairy cell leukemia and a subgroup of B-cell lymphomas (Lynch et al., 1993), low grade human bladder carcinoma (Fanning et al., 1992), neuroblastoma (Bolen et al., 1985; O'Shaughnessy et al., 1987; Bjelfman et al., 1990), ovarian cancer (Wiener et al., 1999) and non-small cell lung carcinoma (Budde et al., 1994). In the case of colon cancer, Src is activated more frequently than Ras or p53 (Jessup and Gallick, 1993), and undergoes two distinct activations corresponding with malignant transformation of colonocytes (Cartwright et al., 1990) and tumor progression (Talamonti et al., 1991, 1992; Termuhlen et al., 1993). Antisense to Src inhibits growth of human monoblastoid leukemia cells (Waki et al., 1994), K562 human leukemia cells (Kitanaka et al., 1994) and HT-29 human colon cancer cells (Staley et al., 1995). Src activity was reduced in a human ovarian cancer cell line (SKOv-3) by antisense technology. The reduced Src activity in SKOv-3 was associated with altered cellular morphology, reduced anchorage-independent growth, diminished tumor growth and reduced vascular endothelial growth factor mRNA expression in vitro (Wiener et al., 1999). In addition, growth inhibition of colon tumor (Garcia et al., 1991; Novotny-Smith & Gallick, 1992) and neuroblastoma cell lines (Preis et al., 1988) correlate with decreases in tyrosine kinase activity of Src. In a colon adenocarcinoma cell line, HT29, the mRNA expression of vascular endothelial growth factor (VEGF) was decreased in proportion to the decrease in Src kinase activity caused by expression of a Src antisense expression vector. In nude mice, there was a decrease in tumor vascularity in subcutaneous tumors from Src antisense transfectants (Ellis et al., 1998).
Changes in Src activity are associated with changes in the cell cycle (Chackalaparampil & Shalloway, 1988) and alterations in the regulation of Src activity have been associated with neoplasia (Bolen et al., 1985; Bolen et al., 1987; Zheng et al., 1992; Sabe et al., 1992). Inhibition of Src would have the effect of interrupting the signal transduction pathways in which it participates and would thereby reduce the rate of growth of cancer cells. Drugs directed to inhibit the Src family may have the advantage of limited or no systemic toxicity but high specificity for tumors shown to have elevated activity of one or more members of the Src family.
In addition to their potential as anti-tumor agents, Src inhibitors have potential for treatment of osteoporosis, a condition in which bone resorption is increased resulting in weakening of bone. It was shown that mice depleted of the Src gene developed osteopetrosis (Soriano et al., 1991) and that Src is involved with bone resorption (Hall et al., 1994). Herbimycin A, a Src inhibitor, inhibits osteoclastic bone resorption in vivo (Rodan and Martin, 2000).
Excessive tyrosine kinase activity is associated with cancer and autoimmune diseases. Tyrosine kinase inhibitors are currently being studied for use in treatment of hematologic tumors, solid tumors, inflammatory diseases and autoimmune diseases (Sinha and Corey, 1999). Treatments which alter the levels of Fyn in appropriate tissues have been proposed to be effective treatments in alcoholism and autoimmune disease (Resh, 1998). Lck and Fyn play an important role in T cell activation through their association with CD4 and CD3, respectively. Autoimmune disease could by treated by inhibition of T cell activation through Lck and Fyn (Sinha and Corey, 1999). In allergic/immunological diseases, development of inhibitors of Lyn, Hck, Lck, Fgr, and Blk are proposed to be useful in treatment of allergic diseases, autoimmunity, and transplantation rejection (Bolen and Brugge, 1997). Some members of the Src family are targets for treatment or prevention of allergic responses; for example, Lyn is indispensable for mast cell-mediated allergic responses (Hibbs and Dunn, 1997). Lyn plays a role in B cell receptor and IgE receptor signal transduction. Inhibition of Lyn may provide a treatment for anaphylaxis or allergy. Lyn-deficient mice are unable to experience anaphylaxis (Sinha and Corey, 1999). The Src-family of tyrosine kinases plays a critical role in blood cell function. Many members of the Src-family of tyrosine kinases are found exclusively or primarily in blood cells. These members are Fgr, Blk, Lck, Lyn, and Hck. Defects in the Src-family of tyrosine kinases have been observed in patients with hematologic disease. Inhibitors of Src kinases have been shown to block leukemic cell growth (Corey et al., 1999).
The level of Fyn, a Src family tyrosine kinase, is up-regulated in Alzheimer's Disease. The phosphorylation by Fyn of the microtubule-associated protein, tau, affects the ability of tau to bind to microtubules. Abnormally phosphorylated tau is found in the neurofibrillary tangles of Alzheimer's Disease. It is also thought that the Aβ peptide in senile plaques activates tyrosine kinases (Lee et al., 1998). Src has been demonstrated to regulate the NMDA receptor (Yu and Salter, 1999). Therefore, the neuronal Src family members may be prime targets for treating CNS disorders including, but not limited to, Parkinsons Disease and chronic pain (Wijetunge et al., 2000). Neuronal Src kinase activity is increased in hippocampal slices treated with a potassium channel blocker in Mg2+-free medium to induce epileptiform discharges. The frequency of the epileptiform discharges is decreased by the addition of an inhibitor of the Src family of tyrosine kinases. Therefore, the Src family may provide a key target for treating epilepsy and other disorders related to NMDA receptor function (Sanna et al., 2000).
Herpesviridae, papovaviridae, and retroviridae have been shown to interact with non-receptor tyrosine kinases and use them as signaling intermediates. The HIV-1 Nef protein interacts with members of the Src family of tyrosine kinases. Nef mediates downregulation of CD4 membrane expression, modification of T-cell activation pathways, and increases virus infectivity (Collette et al., 1997). The HBx protein of the hepatitis B virus is essential for infection by hepadnaviruses and activates Ras by activating the Src family of tyrosine kinases. The activation of Ras is necessary for the ability of the HBx protein to stimulate transcription and release growth arrest in quiescent cells (Klein and Schneider, 1997). Activity of the Src family of tyrosine kinases is altered by association with viral proteins such as mouse and hamster polyomavirus middle-T antigens, Epstein-Barr virus LMP2A, and herpesvirus saimiri Tip (Dunant and Ballmer-Hofer, 1997).
Src family tyrosine kinases have also been implicated in bacterial infection. For example, Pseudomonas aeruginosa is a bacteria that causes opportunistic infections in humans. Both its invasion and cytotoxicity involves the Src family (Evans et al, 1998). Likewise, gonorrhea is caused by the bacterium Neisseria gonorrhoeae and its invasion requires the activity of the Src PTK family (Hauck et al., 1998).
Potential sites for targeting inhibitors of Src are the SH2 and SH3 domains (Waksman et al., 1992; Luttrell et al., 1994), the phosphoryl transfer site (SH1 domain), or other unknown sites on the enzyme. Compounds binding to SH2 and SH3 domains would block the protein-protein interactions and the recruitment of other signal transduction proteins mediated by these domains. Inhibitors of the present invention are targeted to the phosphoryl transfer site (SH1 domain), i.e., the active site. Active-site directed inhibitors could be targeted to the ATP binding site, the protein substrate binding site, or both (bisubstrate analogues).
Several “small molecule” inhibitors of PTKs isolated from natural products have been identified, such as lavendustin A, piceatannol, erbstatin, quercetin, genistein, herbimycin A, etc. (reviewed by Chang and Geahlen, 1992; Burke, 1992). The polyhydroxylated phenyl and styryl groups of erbstatin and piceatannol, and the salicyl group of lavendustin A have been identified as pharmacophores and several groups have carried out structure activity studies of molecules possessing these features (Burke, 1993, 1994; Chen et al., 1994; Cushman et al., 1991a,b,c; Dow et al., 1994; Fry et al., 1994; Maguire et al., 1994; Thompson et al., 1994). The small molecule inhibitors tend to compete with ATP binding and show low specificity for individual PTKs. Typically, these inhibitors have IC50 or Ki values in the low μM range for a variety of PTKs, the exceptions being PD 153035 (4-(3-bromophenylamino)-6,7-dimethoxyquinazoline) of Fry et al., (1994) which has a Ki of 5 pM for EGFR kinase activity, and CAQ (4-(3-chlorophenylamino)-quinazoline), which has a Ki value between 16 and 32 nM (Ward et al., 1994).
Prior to the present invention, few potent small-molecule inhibitors of the Src family possessed suitable pharmacokinetics, affinity, or specificity to serve as effective treatments for disease (Zhu et al., 1999; Sun et al., 2000; Missbach et al., 2000). Therefore, the inventors of the present invention have identified a number of thienopyrimidine-based inhibitors of the Src family that are suitable to act as pharmaceuticals.